Plasmid Control of Symbiotic Properties In

PLASMID CONTROL OF SYMBIOTIC PROPERTIES IN

Rhizobiumfredii

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN MICROBIOLOGY

AUGUST, 1986

Maria Luz Cases

Thesis Committee

B. Ben Bohlool, Chairman

Leslie R. Berger

Francoise M. Robert

We certify that we have read this thesis and that in our opinion,

it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Microbiology.

ACKNOWLEDGEMENTS

I would like to express my gratitude to Dr. Ben Bohlool for the opportunity to do research under his tutelage and financial support. I would also like to thank the members of my committee for their constructive suggestions in the preparation of this thesis.

I am indebted to Mark Kingsley for his valuable contributions in all aspects of this research by his technical guidance, helpful comments find friendship. I would also like to acknowledge Dr. Duane Bartholomew of the Department of Agronomy for the use of the growth chambers.

Lastly, I would like to say a very special thank you to my parents and family for their support and encouragement, and to my best friend, Thomas George, for his help, patience, and understanding.

The research was funded in part by grants 58-9AHZ-2-670 from

the U. S. Department of Agriculture and DAN-5542-G-SS-2095-00 from the U. S. Agency for International Development.

ABSTRACT

Large indigenous plasmids are a common feature in members of the genus Rhizobium, and their involvement in the control of symbiotic functions has been established. Studies on these rhizobial plasmids, called pSym plasmids, make up much of what is understood about the molecular and genetic basis of the N2-fixing symbiosis. The soybeans, generally nodulated by slow-growing rhizobia, are an economically important crop but genetic studies about this group of rhizobia have made little progress because of their slow growth rate and the absence of identifiable plasmid-associated symbiotic functions. An approach to understanding the symbiosis in the soybean system is to use as a genetic model, R.fredii, the fast-growing rhizobia that nodulate soybeans.

In this study, five strains of R.fredii were examined for the presence of indigenous plasmids. To determine if symbiotic functions are controlled by genes on these plasmids, the strains were subjected to plasmid-curing treatments. The effect of a pSym plasmid from a heterologous species on R.fredii gene function was studied by the introduction of the R.leguminosarum pSym plasmid, pJB5JI.

The results of this study show that high molecular weight plasmids that are involved in determining symbiotic functions, as well as cryptic plasmids, are an integral part of the genetic make-up of R.fredii. These strains can receive and maintain p7B5JI, a pSym plasmid from another Rhizobium species. However, the pJB5JI plasmid genes are not expressed in the R.fredii genetic background. The introduction of the plasmid did not enable any of the R.fredii transconjugants to nodulate peas, nor did it restore the ability of the plasmid-cured transconjugants to nodulate soybeans. The presence of the plasmid affected the expression of the R.fredii symbiotic genes, resulting in different levels of symbiotic effectiveness.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... 3

ABSTRACT ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

CHAPTER 1.INTRODUCTION ...... 9

CHAPTER 2. MATERIALS AND METHODS ...... 22

Bacterial Strains ...... 22 Maintenance of Cultures ...... 22 Assessment of Culture Purity and

identification of strains ...... 22

Plasmid Profile Analysis ...... 25

Development of Mutants ...... 26

Bacterial Matings ...... 28

Plant Infection Tests ...... 29

CHAPTER 3. RESULTS ...... 32

Plasmid Profiles ...... 32

Isolation of Plasmid-Cured Mutants ...... 38

Transfer, Expression and Maintenance

of pSym Plasmid ...... 41

CHAPTER 4. DISCUSSION ...... 58

LITERATURE CITED...... 66

LIST OF TABLES

TablePage

1Characteristics and sources of

Rhizobium strains used in the study ...... 23

2Acid production and FA reactions of

R.fredii and R.leguminosarum strains ...... 33

3Intrinsic antibiotic resistance patterns

of R.fredii and R.leguminosarum strains ..... 34

4Phage-typing patterns of R.fredii and

R.leguminosarum strains ...... 35

5Symbiotic properties of parent and plasmid-

cured R.fredii strains on soybeans ...... 42

6Frequency of natural kanamycin resistance

in R.fredii strains ...... 44

7Frequency of transfer of kanamycin resistance

marker in crosses between R.leguminosarum 6015

(pJB5JI) and R.fredii ...... 45

8Presence or absence of the plasmid pJB5JI

and symbiotic properties of R.fredii

transconjugants...... 46

9Frequency of transfer of kanamycin resistance

marker in backcrosses between R.fredii

transconjugants and R.leguminosarum 6015 ..... 50

10Summary of the pJB5JI donor, plasmid

content, and symbiotic properties of

R.leguminosarum 6015 transconjugants ...... 54

11Nodulation and nitrogen fixation by

isolates of R.fredii transconjugants after

first passage on Peking soybeans ...... 55

12Nodulation and nitrogen fixation by

isolates of R.fredii transconjugants after

second passage on Peking soybeans ...... 57

LIST OF FIGURES

FigurePage

1Agarose gel electrophoresis of plasmid

DNA from R.fredii ...... 37

2Plasmid profiles of R.fredii cured

mutants and parent strains ...... 40

3Plasmid profiles of USDA 205-A05

transconjugants and reference strains ...... 49

4Plasmid profiles of R.leguminosarum

6015 and its transconjugants ...... 52

CHAPTER 1

INTRODUCTION

The rhizobia are Gram-negative soil bacteria belonging to the genus Rhizobium which fix nitrogen in a symbiotic association with legumes. Establishment of the N2-fixing symbiosis starts with the colonization, recognition and invasion of the plant root by free-living rhizobia in the soil, followed by a series of steps that result in the formation of a nodule. It is in these nodules that the rhizobia differentiate into bacteroids and reduce atmospheric nitrogen to ammonia with the enzyme nitrogenase.

In the symbiosis, both the plant host and the bacterial endosym-biont play particular roles that are regulated by gene expression. The frequency with which certain symbiotic properties such as infectiveness and effectiveness were lost, and the stimulation of this loss by treatments known to affect plasmids has led to the suggestion that symbiotic properties are plasmid-borne.

Indigenous plasmids in rhizobia

The presence of plasmids with molecular weights higher than 100 megadaltons (Mdal) is a general feature in the family Rhizobiaceae. This has been correlated with some biological effects of the bacterial symbiont on the plant, including the induction of crown gall by Agrobacterium and the establishment of N2-fixing symbioses by Rhizobium and Bradyrhizobium. In rhizobia, the plasmids have also been found to bear genes for the production of the melanin pigment (Beynon, et al., 1980), bacteriocins (Hirsch, 1979), cell wall polysaccharides (Prakash, 1980) and uptake hydrogenase (Brewin et al., 1980c). However, the function of most of the DNA in these plasmids remains unknown.

Strains of both the fast-growing Rhizobium and the slow-growing Bradyrhizobium typically harbor one to several plasmids. A great percentage of their DNA is in the form of large, low copy number (1-3 per cell) plasmids, representing at least 5 to 20 per cent of the total genomic information (Beringer et al., 1980).

In terms of physical evidence, many indigenous plasmids were isolated by alkaline denaturation-phenol extraction and visualized by gel electrophoresis (Casse et al., 1979; Hirsch et al., 1980; Nuti et al., 1977; Prakash et al., 1980). The larger megaplasmids were detected by the more sensitive Eckhardt direct-lysis method of gel electrophoresis (Denarie et al., 1981; Eckhardt, 1978; Rosenberg et al., 1982;).

Large plasmids were found in Rhizobiumleguminosarum, Rhizobiumtrifolii, Rhizobiumphaseoli, and Rhizobiummeliloti (Beynon et al., 1980b; Casse et al., 1979; Denarie et al., 1981; Hirsch et al., 1980; Nuti et al., 1977; Prakash, 1980). In R.meliloti, different isolation procedures revealed two classes of plasmids. One class consisted of medium range 90- to 200-Mdal plasmids (Casse et al., 1979) and a second class consisted of plasmids with sizes greater than 300 Mdal, referred to as megaplasmids (Banfalvi et al., 1981; Denarie et al., 1981; Rosenberg et al., 1981). By means of electron microscopy, Burkhardt and Burkhardt (1984) estimated their size to be 1000 Mdal.

In the case of slow-growing rhizobia, Gross et al. (1979) examined Bradyrhizobiumjaponicum isolated from alkaline soils and observed large plasmids. Other strains from various geographical origins ware examined by Masterson et al. (1982) and all of those examined contained at least one large plasmid, with sizes ranging from 118 to 1915 Mdal. They concluded that the presence of plasmids is a consistent feature of most B.japonicum strains.

Evidence has been presented that in the endosymbiotic bacteroids, the large plasmids are extensively transcribed. Krol et al. (1980) studied R.leguminosarum and found that in contrast to the bacteroids, there was no detectable transcription of the plasmid in broth cultures, showing that under these conditions, plasmid genes are not expressed. They suggested that the large rhizobium plasmids contain not only the genes that control N2-fixation but also the genes which are functional during the differentiation of bacteria into bacteroids.

Plasmid involvement in symbiotic nitrogen fixation

Physical and genetic studies have now established that, at least in fast-growing Rhizobium species, the majority of the genes that control symbiotic functions such as host-range specificity, nodulation, and N2-fixation are usually on a plasmid referred to as the pSym plasmid.

In several fast-growing Rhizobium species, genes controlling symbiotic functions have been localized on large plasmids ranging in size from 130 Mdal in R.leguminosarum (Hirsch et al., 1980) to more than 450 Mda1 in R.meliloti (Denarie et al., 1981). In slow-growing rhizobia such as B.japonicum, plasmids controlling symbiotic functions have not been identified and the symbiotic genes are presumed to be located on the chromosome.

Host range specificity

Host selectivity at the early stages of infection is a striking feature of the rhizobia-plant interaction. With one known exception (Parasponia), the rhizobial plant hosts are members of the family Leguminosae, and within this family, groups of plants are nodulated by particular Rhizobium species. There is evidence that the pSym plasmid carries genes determining host-range specificity (hsn).

Brewin et al. (1981) reviewed the role of Rhizobium plasmids in host specificity, and concluded that, at least for the three closely related species, R.leguminosarum, R.trifolii, and R. phaseoli, host-range is a plasmid-determined trait.

Plasmid-controlled host specificity in R.leguminosarum was first described by Johnston et al. (1978). In the study, a R.leguminosarum plasmid, pJB5JI, was transferred to R.trifolii and R.phaseoli. All transconjugants were capable of forming nodules on peas in addition to their normal hosts. By cloning the nodulation genes of the plasmid, Downie et al. (1983) showed that host specificity is determined by a DNA sequence of no more than 10 kilobases (kb).

In R.trifolii, mutagenesis of host-specific nodulation genes in the pSym plasmid by the transposon Tn5, a short segment of DNA coding for the antibiotic kanamycin and which is capable of inserting within a genome, resulted in mutants with altered host-range ability. The mutants showed either poor nodulation on clover or none at all (Djordjevic et al., 1985). A 14-kb fragment of the pSym plasmid was mobilized and conferred clover-specific nodulation on another strain of R.trifolii that was cured of the pSym plasmid, as well as on Agrobacteriumtumefaciens, a member of another genus in Rhizobiaceae (Schofield et al., 1984).

Particular host-range characteristics within a cross inoculation group were also found to be plasmid-controlled. The strain R.leguminosarum TOM, which nodulates the primitive pea cultivar Afghanistan, was able to transfer this trait at low frequencies to a strain that does not normally nodulate this cultivar. The transfer of cultivar specificity was associated with the transfer of a 160-Mdal plasmid designated pRL5JI (Brewin et al., 1980).

Other reports gave evidence for the presence of host-range determinants on pSym plasmids (Appelbaum et al., 1985; Beynon et al., 1980; Djordjevic et al., 1983; Hooykaas et al., 1981; Kondorosi et

al., 1982; Morrison et al., 1983).

Nodulation

Successful interaction of a rhizobia with their plant host results in the development of an organized structure, the root nodule. The early stages of nodule formation include root hair colonization and adhesion, root hair curling, infection thread development, cortical cell multiplication, release of rhizobia in the host cells, and proliferation of bacteria within the plant cells. The later stages encompass events such as bacteroid differentiation, host cell enlargement, and the start of nitrogen fixation (Verma and Long, 1983).

The role of plasmids in the control of nodule formation has been demonstrated by studies on plasmid-cured mutants, transposon mutagenesis, and plasmid transfer experiments.

The association of plasmid loss and a non-nodulatiog (Nod-) phenotype provided early evidence of plasmid involvement in nodulation. Higashi (1967) found that the ability of R.trifolii to nodulate clover was lost at high frequency following treatment with acridine orange, a chemical agent known to cause the elimination of plasmids (Clowes et al., 1965; Parijkaya, 1973).

Zurkowski and Lorkiewicz (1976, 1978) correlated plasmids in R.trifolii with nodulation, by showing that Nod- mutants resulting from a prolonged treatment at high temperature were due to either loss of plasmid DNA or internal deletions in the plasmid. Zurkowski (1982) reported that at high temperature, DNA synthesis stops while protein synthesis continues, leading to the formation of enlarged cells, and the loss of the plasmid during cell division. The transfer of the plasmid into the Nod- strains which had lost the ability to attach to the root hair surface, converted them to a Nod+ phenotype (Zurkowski, 1981). The absence of the symbiotic function that was associated with plasmid loss and its restoration with plasmid transfer, point to the involvement of the plasmid in the nodulation process.

Similarly, prolonged heat treatment of R.leguminosarum resulted in Nod- strains that exhibited modified surface properties (Prakash et al., 1980). The mutants lost the ability to agglutinate in the presence of pea lectin, a plant protein that recognizes and binds with specific carbohydrate components of the bacterial cell surface. Lectin binding is a function that has been correlated with host specificity (Bohlool and Schmidt, 1974; Dazzo and Hubbel, 1975).

In R.meliloti, the early functions in the infection process are reported to be on a megaplasmid (Rosenberg et al., 1981). Previously, Palomares et al. (1978) had shown that in R.meliloti, extrachromosomal DNA was responsible for polygalacturonase, a key enzyme originally thought to be involved in the early infection process (Ljunggren and Fahraeus, 1961). Long et al. (1982) cloned the nod genes which complement the nodulation defect of a Nod-R.meliloti mutant and assigned the nodulation function to a region of the 8.7-kb EcoRI fragment on the megaplasmid.

Although the nodulation process is characterized by a high degree of specificity, some of the genes involved in the early steps of nodulation were found to be conserved and common across different rhizobia. A proposed genetic model is that there is a core of nodulation-specific genes that are essentially the same in different Rhizobium species and that host range is determined by ancillary host range genes (Downie et al., 1983).

Kondorosi et al. (1984) identified DNA regions of a R.meliloti megaplasmid carrying nod genes involved in root hair curling, an early step in nodule formation. The genes, referred to as the common nod genes, are active in a wide range of plant hosts and mutations in these can be complemented by pSym plasmids from other Rhizobium species such as R.leguminosarum. The genes nodABCD, which are clustered in the pSym plasmid have been identified in several species of Rhizobium (Banfalvi et al., 1981; Djordjevic et al., 1985; Fischer et al., 1985).

The functional conservation of nod genes in the fast-growing strains may also extend to the slow growers. When DNA fragments from Bradyrhizobium sp. (Parasponia) were introduced into a Nod-R.meliloti strain, nodulation ability was restored (Marvel et al., 1984). Noti et al. (1985), using a DNA region from Bradyrhizobium sp. (Vigna), reported similar findings.

Nitrogen fixation

Johnston et al. (1978) demonstrated that the transfer of a R.leguminosarum plasmid into a Fix- strain restored its normal symbiotic function, implying that the genes that control the ability to fix nitrogen are located on plasmids. Furthermore, the transfer of the R.leguminosarum bacteriocinogenic plasmid pRLlJI into symbiotic mutants, including nodulation-deficient ones restored them to Fix+ phenotypes (Brewin et al., 1980a). The location of the genes involved in N2-fixation was confirmed by hybridization with a recombinant DNA clone carrying the nitrogenase (nif) genes from Klebsiellapneumoniae. The K.pneumoniae nifD and nifH genes, which encode two of the three nitrogenase subunit polypeptides, hybridizes to DNA restriction fragments of many diverse N2-fixing bacteria, indicating the conservation of the nif genes at the DNA sequence level (Ruvkun and Ausubel, 1980a). Hybridization experiments have established the presence of nif genes in R.leguminosarum (Nuti et al., 1979), R.phaseoli (Hombrecher et al., 1981), R.trifolii (Hooykaas et al., 1981; Schofield et al., 1985), R.meliloti (Banfalvi et al., 1981; Rosenberg et al., 1981), and a fast-growing cowpea Rhizobium (Morrison et al., 1983). Homology studies between the pSym plasmids of diverse fast-growing Rhizobium species showed that a specific DNA sequence which carries the structural genes for nitrogenase is highly conserved in R.leguminosarum, R.trifolii, and R.phaseoli (Prakash et al., 1981).

Slow-growing rhizobia, including B.japonicum have not been shown to carry nif sequences on plasmid DNA (Masterson et al., 1982).

Linkage of genes

The genes for nodulation and N2-fixation as well as for other functions are linked on a plasmid in most Rhizobium strains. In many cases, the pSym plasmid found to bear nif sequences was also known to encode for nodulation and host-range specificity, and for other functions.

Tn5 insertions in a R.leguminosarum plasmid (Buchanan-Wollaston et al., 1980) and in a R.meliloti megaplasmid (Meade et al., 1982) could produce both Nod- and Fix- phenotypes. In studies of the R.meliloti megaplasmid, it was shown that in a large number of spontaneous Nod- mutants, a deletion of the megaplasmid occurred with the concommitant loss of the sequence homologous to nif. By analyzing such deletions, Banfalvi et al. (1981) and Rosenberg et al. (1981) deduced close linkage of the nod and nif loci. Physical and genetic data have confirmed that the nod genes are located within 30 kb of the nif genes on the megaplasmid (Long et al., 1982).

Hybridization experiments showed that nodulation and nitrogen fixation genes are found in the same plasmid in R.leguminosarum (Hombrecher et al., 1981), R.phaseoli (Lamb et al., 1982), R.trifolii (Hooykaas et al., 1981) and a fast-growing cowpea Rhizobium (Morrison et al., 1983). The molecular linkage map of the nitrogenase and nodulation genes was constructed in R.trifolii by Schofield et al. (1983). Hooykaas et al. (1981) reported that the R.trifolii pSym plasmid not only determines host specificity for clover, but also controls other steps in nodulation and N2-fixation.

In crosses between R.leguminosarum and R.trifolii, host-range specificity is cotransferred with other nodulation loci (Djordjevic et al., 1983; Downie et al., 1983; Hombrecher et al., 1984; Schofield et al., 1984).

Close linkage with the symbiotic genes on the pSym plasmids is shown by genes that control other functions. Beynon et al., (1980b) observed that spontaneous plasmid deletions eliminated both melanin producticn and the ability to nodulate Phaseolus beans and suggested that the genes involved in the control of both functions are on a single plasmid. Brewin et al. (1980c) identified a R.leguminosarum plasmid which not only carried nif and nod genes but also specified the genes controlling the production of uptake hydrogenase, an enzyme that catalyzes the oxidation of the hydrogen liberated during nitrogen reduction.